The invention relates to a system for performing a assay of a cell sample to provide an accurate quantitative analysis of a characteristic of the cells which have been sampled. More particularly, the invention is directed to a system which receives images of stained cells and enhances the cell images prior to further processing to determine the proliferation index of the enhanced cell images.
One of the problems which faces pathologists in their clinical practice is that of determining whether a cell sample taken from a patient during a biopsy procedure or the like is benign or malignant. Although a surgeon may have a good intuition about the type of tissue mass which he has removed, nevertheless he must confirm his preliminary diagnosis with a histological examination of the cell sample removed from the patient. The histological examination entails cell staining procedures which allow the morphological features of the cells to be seen relatively easily in a light microscope. A pathologist after having examined the stained cell sample, makes a qualitative determination of the state of the tissue or the patient from whom the sample was removed and reaches a conclusion as to whether the patient is normal or has a premalignant condition which might place him at risk of a malignancy in the future or has cancer. While this diagnostic method has provided some degree of predictability in the past, it is somewhat lacking in scientific rigor since it is heavily reliant on the subjective judgement of the pathologist.
Attempts have been made to automate the cellular examination process. In U.S. Pat. No. 4,741,043 to Bacus for Method and Apparatus for Image Analyses of Biological Specimens, an automated method and a system for measuring the DNA of cells are disclosed which employ differential staining of the DNA in cell nuclei with a Feulgen Azure A stain and image processing. While the system provides an accurate assay of the cellular DNA its predictive power for cell replication, a key indicator of the presence of cancer, could be improved.
It is well known that cells follow a replication cycle; for a further discussion of the cycle reference may be made to Pages 330-336 of McGraw-Hill Encyclopedia of Science and Technology, 6th Edition, 1987. Most somatic cells of an adult human replicate at a relatively slow rate, only rapidly enough to replace cells shed by the body and lost to normal cellular wear and tear. At any instant, most of those somatic cells are in the GO- or resting phase of the replication cycle. When they leave the resting phase they enter the Gl or first gap phase but are not yet producing extra DNA. Upon becoming committed to the S-phase, however, they do produce other material such as proliferation substances e.g. cyclin and other S-phase proteins. The cells in the synthesis or S-phase are actively synthesizing DNA and produce double the amount of DNA normally contained in the cell nuclei in preparation for mitosis or division of the cell nuclei during cell replication. A normal human somatic cell contains 23 chromosome pairs and is in the diploid state. The diploid state is also referred to as the 2N state. At the time of replication the number of chromosome pairs increases to 46, double the normal amount in antic of cell division. The chromosome state immediately before replication is referred to as the 4N state. The cells then enter the second gap phase or G2 phase in which little or no DNA is synthesized. Following the G2 phase is the mitosis or M-phase in which the cells themselves divide. If the cells are actively proliferating they may reenter the G1 phase.
Although DNA analysis may be adequate for estimating the number or proportion of proliferating cells in normal cells or tissue, it should be appreciated that this is not the case with malignant cells, the very ones for which it often is important to know the extent of proliferation. This is because malignant cells often have increased amounts of DNA, even in the G0 phase, due to increased chromosome content, and often increased of chomosomes. Therefore, it is impossible to conclude with certainty from a DNA analysis that a particular cell, e.g. one having 1.5 times the normal DNA content, is a malignant cell with additional chromosomes, or chromosome parts, or is a normal cell which is halfway through the S-phase having only replicated one-half the DNA necessary for cell division. Thus it is clear that an analysis method independent of DNA, utilizing other markers, such as variously produced proteins associated with S-phase proliferation and the cell division process, has many advantages
It should also be appreciated that quantitating on a cellular proliferation index has previously been performed by counting the numbers of cells in a cell sample carrying an indicator or stain for a proliferation substance. For instance, a well known method of determining the proliferation index is to stain the cells with an immunofluorescent dye which binds to cyclin and manually count the fluorescent and non-fluorescent stained cells to determine the proportion of cells having proliferation substance.
Another method of determining the proliferation index of cells is the grain counting method; for a further discussion of this grain counting method reference may be made to Pages 107-112 of The American Journal of Pathology, Volume 134, No. 1, January, 1989. In that method, tritiated thymidine is added to a cell culture growth medium. Proliferating cells take up the tritiated thymidine and incorporate it into DNA being synthesized in the cells. The cells are then fixed and placed in proximity with a photographic emulsion. Decay products of the tritium expose portions of the emulsion. The exposed portions may be visualized as grains by photographic development processes. Cells with overlying grains and with non-overlapping grains, are then counted to determine the proliferation index. One of the drawbacks of this method lies in the fact that it is very time consuming. It is necessary that the cells be harvested alive and kept alive long enough to take up the tritiated thymidine. The cells must then be fixed and held in proximity with the emulsion in order to expose it. Since relatively low intensities of radiation may emanate from the cells, it may take days or even weeks to obtain a latent image on the emulsion, which must then be developed. In the meantime, the patient's disease may be progressing.
One of the drawbacks of the prior art methods is that they are prone to human error due to the tedium of counting the cells on a microscope slide under high magnification. Often the people examining the slides only are able to estimate the relative number of cells which show a positive result for proliferation substance.
The prior imaging systems have also suffered from the problem that while they usually accurately identify the images of cell objects in an image being processed they do not always accurately identify boundaries of the cell objects being evaluated. This may be a problem when an assay is being performed on the cell objects on the basis of their image areas.
The prior art methods of quantitatively analyzing the cell samples for proliferation substances could not be automated simply. This is because it is necessary to determine a baseline value for the total number of cells examined as opposed to the number of cells which have proliferation substance. In order to make this type of evaluation an automatic system must be able to recognize what constitutes a cell or a cell nucleus. In order to solve this baseline recognition problem the instant invention employs separate stains for the cell nuclei and the proliferation substances. In addition, the stains are separated spectrally so that they can be readily distinguished by optical filters which are compatible with them. The optical separation of the two components to be measured makes the subsequent analysis of the cell images more convenient to automate.
A similar difficulty is encountered in an image analysis based on cell object areas when cell objects images overlap, touch or otherwise share contiguous areas. In that case, what is actually a double or triple object image may not be tallied properly resulting in an inaccurate result or conclusion.